Project Summary About 15-20% of breast cancer patients fall under the basal-like category, which represent a diverse subtype that is characterized by tumors that are more aggressive conferring poor prognosis. Deaths from these carcinomas result from metastatic spread of the disease to distant sites and from therapeutic resistance, which results in the relapse of cancers into more aggressive forms that are difficult to contain. Both these properties are attributed to their cellular heterogeneity that arises through various mechanisms including clonal evolution and the presence of cancer stem cells (CSCs). The epithelial-to-mesenchymal transition (EMT) is one program that we have shown to be responsible for the generation of cells that have CSC-like properties. Our current proposal aims to induce differentiation of these CSCs through the induction of a mesenchymal-to- epithelial transition (MET). To do this, we carried out a screen to identify compounds that are capable of inducing the transcription of E-cadherin, a hallmark of the epithelial/non-CSC state, in cells that are more mesenchymal/CSC-like. Through this screen we identified Forskolin, an activator of cAMP, to be able to induce E-cadherin transcription and a reversion of the mesenchymal/CSCs to a more benign epithelial state. Through this proposal we aim to uncover the mechanism by which cAMP-elevating agents are able to induce an MET by complete characterization of the essential downstream components of signalling, namely Protein Kinase A (PKA) and its downstream substrates. We also aim to understand the epigenetic reprogramming that occurs following activation of PKA through the functions of the histone-modifying enzyme PHF2. Both these components will address the mechanisms by which CSCs can be differentiated to a state that renders them highly sensitive to treatment with chemotherapeutic agents such as doxorubicin. Despite over two decades of research, our ability to specifically target CSCs is still lacking. Through the study of Phosphodiesterases and G-Protein Coupled Receptors (GPCRs) that modulate cAMP levels, I aim to develop ways to specifically induce differentiation of CSCs that can be translated for therapeutic utility.